Intravenous administration of recombinant tPA remains the only FDA-approved and the most beneficial proven intervention for emergency treatment of stroke. However, short treatment time window, hemorrhagic transformation, poor thrombolytic perfusion rate, and neurotoxicity comprise the major limitations to the application. In this proposal, we hypothesize that recombinant Annexin A2 protein (rA2) will lower the required dose of tPA for reperfusion, while enhancing thrombolytic efficacy, and attenuating intracerebral hemorrhagic transformation. By doing so, it will prolong therapeutic time windows and improve long-term outcomes. The molecular basis for our idea is that annexin A2 accelerates the generation of clot-dissolving plasmin by assembling a fibrinolytic complex with tPA and the plasmin precursor plasminogen. Our pilot data are promising. We now propose three aims to investigate the mechanisms involved in this new combination therapy. In Aim 1, we will study thrombolytic efficacy of rA2 plus low dose tPA combination treated at 4 hrs after focal embolic stroke in rats with MR angiography to determine recanalization and reocclusion, MRI CBF to map cerebral blood flow; and MRI measurements of cerebral blood volume (CBV), microvascular volume (MVV), and vessel size index (VSI) to quantify vascular responses to thrombolytic agents and vasodilators. In Aim 2, we will investigate effects of rA2 plus low dose tPA combination therapy on neurovascular matrix proteolysis and BBB leakage treated at 4 hrs after focal embolic stroke of rats. The development of brain infarction, edema and hemorrhagic transformation will be measured and correlated with temporal profiles of MMP-9 activity, proteolytic degradation of extracellular matrix components and BBB leakage. In Aim 3, we will assess long term outcomes of rA2 plus low dose tPA combination treated at 4 hrs after stroke. Rats will be followed over 4 weeks with a battery of behavioral tests. At the end, brains are removed for H&E lesion size measurement, cerebral vessel density and tissue recovery factors VEGF, Ang-1 expression will be examined. Low-dose tPA plus rA2 will be compared with rA2 alone, low-dose and high-dose tPA alone, and vehicle saline control. If successful, these proposed experiments should provide new insight of how rA2 plus low dose tPA improve tPA stroke treatment and may ultimately yield a novel combination approach to optimize tPA-based thrombolytic stroke therapy. We believe our proposal should be directly relevant for PA-07-233, Multidisciplinary Translational Research in Critical Care (R01). PUBLIC HEALTH RELEVANCE: Intravenous administration of tPA is the only FDA-approved emergency treatment for ischemic stroke. Mainly because the risk of brain bleeding, only 2-5% stroke patients receive tPA in USA. Here we propose a new combination therapy of annexin A2 plus tPA, it will generate more beneficial thrombolytic enzyme plasmin with much lower tPA dose. By doing so, the combination will enhance clot lysis for blood flow restorage and reduce the risk of bleeding. These effects should prolong treatment window and improve long-term outcomes.